Antitumor antibiotic BMY-41219

ABSTRACT

A new antitumor antibiotic designated BMY-41219 is produced by fermentation of Saccharothrix aerocolonigenes ATCC 39243. The BMY-41219 antibiotic exhibits antitumor activity.

This application is a continuation of application Ser. No. 07/764,114,filed Sep. 23, 1991, now abandoned, which is a continuation ofapplication Ser. No. 07/489,431 filed Mar. 6, 1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to the production of an antitumor antibioticdesignated herein as BMY-41219 and to a process for the preparation,isolation and purification of BMY-41219.

2. Background Art

U.S. Pat. Nos. 4,487,925 and 4,552,842 disclose the anti-tumor agentdesignated rebeccamycin, and the 5'-methyl and 5', 2', 3",6"-tetraacetate derivatives thereof, and a process for producing thesame agent by cultivating a rebeccamycin-producing strain of Nocardiaaerocolonigenes, preferably Nocardia aerocolonigenes ATCC 39243, or arebeccamycin-producing mutant thereof in an aqueous nutrient mediumcontaining assimilable sources of carbon and nitrogen under submergedaerobic conditions until a substantial amount of rebeccamycin isproduced. Recently Nocardia aerocolonigenes ATCC 39243 was reclassifiedas Saccharothrix aerocolonigenes ATCC 39243 (Bush et al., J. Antibiotics40: 668-678, 1987).

It was found that when the rebeccamycin-producing culture wassupplemented with L-tryptophan, the production of a minor analog ofrebeccamycin was significantly enhanced. This analog of rebeccamycin wassubsequently isolated and designated as BMY-41219.

SUMMARY OF THE INVENTION

The present invention provides a method for the production of an analogof the antitumor agent designated rebeccamycin (Formula I) in thefermentation of Saccharothrix aerocolonigenes ATCC 39243. ##STR1## Morespecifically, there is provided a method for the enhanced production ofa rebeccamycin analog of the Formula II (BMY-41219) below, ##STR2## aswell as pharmaceutically acceptable acid addition salts of such analog.

The compound of the Formulas II is produced by supplementing thecultures of a rebeccamycin producing strain of Saccharothrixaerocolonigenes with L-tryptophan.

DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the ¹ H--NMR for the compound of Formula II.

FIG. 1B shows the D₂ O exchange ¹ H-NMR for the compound of Formula II.

FIGS. 2A and 2B show the FAB-MS for the compound of Formula II (S=matrixNOBA).

FIGS. 2C and 2D show the FAB-MS for the compound of Formula II (S=matrixthiogly.).

FIG. 3 shows the UV spectrum for the compound of Formula II.

DETAILED DESCRIPTION OF THE INVENTION

U.S. Pat. Nos. 4,487,925 and 4,552,842 disclose the production andisolation of the antitumor agent designated rebeccamycin (Formula I)##STR3##

The above-mentioned rebeccamycin compound is the principal component ofthe fermentation of the rebeccamycin producing strain of Nocardiaaerocolonigenes.

It has now been found according to the present invention that thefermentation procedure disclosed in U.S. Pat. Nos. 4,487,925 and4,552,842 can be carried out in the presence of tryptophan to enhancethe production of a analog of rebeccamycin having valuable antitumorproperties. Rebeccamycin is still the principal component of the abovefermentation. The rebeccamycin analog of the present invention has theFormula II below. ##STR4##

Preparation of the Antibiotics

BMY-41219 is produced by cultivating a rebeccamycin producing strain ofSaccharothrix aerocolonigenes, supplemented with L-tryptophan. Thepreferred producing organism is a novel strain of Saccharothrixaerocolonigenes, previously designated as Nocardia aerocolonigenesstrain C38,383-RK2 (ATCC 39243) in U.S. Pat. No. 4,487,925. Recently,this strain was reclassified as Saccharothrix aerocolonigenes (Bush etal., J. Antibiotics 40:668-678, 1987) and is designated herein asSaccharothrix aerocolonigenes strain C38,383-RK2 (ATCC 39243). Thisstrain was isolated from a soil sample collected in Panama. Abiologically pure culture of strain C38,383-RK2 has been deposited withthe American Type Culture Collection, Rockville, Md. and added to theirpermanent collection of microorganisms as ATCC 39243. This culture,designated as C38,383-RK2, is also maintained as a dormant culture inlyophile tubes and cryogenic vials in the Bristol-Myers Squibb Co.Pharmaceutical Research and Development Division Culture Collection, 5Research Parkway, Wallingford, Conn. 06492.

The taxonomic studies on strain C38,383-RK2 (ATCC 39243) have beendescribed in detail in U.S. Pat. No. 4,487,925 and in J. Antibiotics40:668-678, 1987. The strain has been classified as a novel strain ofSaccharothrix aerocolonigenes.

It is to be understood that the present invention is not limited to useof the particular preferred strain ATCC 39243 or to organisms fullyanswering its description. It is especially intended to include otherrebeccamycin producing strains or mutants of the described organismwhich can be produced by conventional means such as x-radiation,ultraviolet radiation, treatment with nitrogen mustards, phage exposureand the like.

In practicing the present process, a rebeccamycin-producing strain ofSaccharothrix aerocolonigenes, having the identifying characteristics ofstrain C38,383-RK2 (ATCC 39243), or a mutant or variant thereof, iscultivated in a conventional aqueous nutrient medium supplemented withL-tryptophan. The organism is grown in a nutrient medium containingknown nutritional sources for actinomycetes. Thus, the organism is grownin a nutrient medium containing an assimilable carbon source such assucrose, lactose, glucose, rhamnose, fructose, glycerol or solublestarch. The medium should also contain an assimilable nitrogen sourcesuch as fishmeal, peptone, peanut meal, cottonseed meal, corn steepliquor, amino acids or ammonium salts. Nutrient inorganic salts can alsobe incorporated in the medium so as to provide sodium, potassium,ammonium, calcium, phosphate, sulfate, nitrate, carbonate and like ions.Trace elements such as copper, manganese, iron, zinc, etc. are added tothe medium if desired, or they may be present as impurities of otherconstituents of the media. For optimal production of BMY-41219, themedium should be supplemented with L-tryptophan. Submerged aerobicconditions are preferably employed for the production of largequantities of antibiotic, although for production of limited amounts,surface cultures and bottles may also be used. The general proceduresused for the cultivation of other actinomycetes are applicable to thepresent invention.

Production of the antibiotic of the present invention can be effected byany temperature conducive to satisfactory growth of the producingorganism, e.g. 18° C. to 39° C. and is conveniently carried out at atemperature of about 28° C. The fermentation may be carried out inflasks or in laboratory or industrial fermentors of various capacity.

When tank fermentation is to be used it is desirable to produce avegetative inoculum in a nutrient broth by inoculating a small volume ofthe culture medium with a slant, a cryopreservative culture or alyophilized culture of the producing organism. After obtaining a viableand active inoculum in this manner, it is transferred aseptically to thefermentation tank charged with production medium for large scaleproduction of the antibiotic of the present invention. The medium inwhich the vegetative inoculum is grown can be the same as, or differentfrom, that utilized in the tank as long as it is such that a good growthof the producing organism is obtained and it is supplemented withL-tryptophan. Further agitation may be provided by a mechanicalimpeller. Antifoam agents such as lard oil or silicone oil may also beadded if needed. Antibiotic production is monitored by high performanceliquid chromatography assay or by conventional biological assay. Ingeneral, optimum production of the antibiotics of the present inventionis achieved after incubation of about 10 days.

Isolation and purification of the so-obtained derivative may be carriedout by conventional chromatographic procedures.

Physical and Chemical Properties

BMY-41219 has following Physical and Chemical Properties:

Description: Yellow-orange amorphous solid

Molecular Formula: C₂₆ H₂₁ N₃ O₇

Molecular weight: 487

Mass Spectrum: FAB (M+1)⁺ ion=488. Reference no. AT0702 and AT0703

Infrared Spectrum: KBr pellet major Bands (cm-1): 2800, 3320, 3200,2925, 1740, 1700, 1670, 1475, 1460, 1405, 1330, 1272, 1240, 1080, 1050,1015, 910, 805, 750, 700, 655

Ultraviolet Spectrum: About 0.6mg/50ml MeOH. Absorption maximum (molarabsorptivity): 236nm (29975), 284nm (17755), 316nm (37418), 404nm(3020).

Analytical HPLC: 10 uG in DMSO. k'--1.7. Column--QBX C18. Eluant--4parts 0.1 M Ammonium Acetate, 3 parts Methanol, 3 parts Acetonitrile.Flow--2.0 ml/min. Detector--315 nm.

200 MHz 1H-NMR: Varian XL-200. Solvent d₆ -DMSO. Observed ChemicalShifts (ppm): 11.70(s, 1H), 9.17(d, 1H), 9.10(d, 1H), 7.97(d, 1H),7.74(d, 1H), 7.57(m,2H), 7.37(m,2H), 6.28(d,H), 5.70(bs, exchangeable),4.17-3.90(m,4H), 3.36(m,2H).

Solubility: Soluble in Dimethyl Sulfoxide, Methanol, andTetrahydrofuran.

Biological Properties

BMY-41219 was tested against the transplanted mouse leukemia P388 todetermine in vivo antitumor activity (Table 1). CDF₁ mice were implantedintraperitoneally (ip) with 10⁶ P388 leukemia cells obtained from DBA/2donor mice bearing this transplantable murine leukemia. The CDF₁leukemic mice were treated ip with either saline (control mice) or dosesof BMY-41219 once at one day post-tumor inoculation. These animals wereobserved daily and their deaths recorded. Average body weight changes(from the day of leukemia implant to the day of last treatment) weredetermined for all groups as a means of reflecting drug toxicity. Theincidence of mice alive in each group on Day 5 post-tumor implant wasrecorded as an additional means of assessing drug toxicity. Notherapeutic result was considered as meaningful if more than one mouseper treatment group had died by Day 5. Each treatment group consisted of6 mice; control groups contained 10 mice. The number of mice, if any,surviving to Day 30 (the last day of the experiment) was also recorded.

Therapeutic efficacy was evaluated by determining the median survivaltime (MST) of BMY-41219 treated mice and comparing it to the MST ofparallel control mice. This comparison was made by dividing the MST ofthe former by the latter and multiplying by 100 to derive a parametercalled the percent T/C value. A percent T/C of ≧125% was considered torepresent a meaningful increase in lifespan and hence an active result.As shown in Table 1, BMY-41219 is active against P388 leukemia at doselevels ranging from 7.5 to 240 mg/kg. The best effect was achieved at adosage of 240 mg/kg and consisted of a %T/C of 170%. Toxicity was notobserved even at the highest dose (240 mg/kg) tested.

                  TABLE 1                                                         ______________________________________                                        Effect of BMY-41219 on P388 leukemia.sup.a                                    (Day 1 treatment)                                                                      Medium            Average No. of Mice                                Dose, ip Survival   %      Weight  Alive on                                   (mg/kg/inj)                                                                            Times (Days)                                                                             T/C    Change (g)                                                                            Day 5 Day 30                               ______________________________________                                        240      17.0       170    0       6/6   0/6                                  120      16.0       160    0.3     6/6   0/6                                  60       14.5       145    0.4     6/6   0/6                                  30       15.0       150    0.1     6/6   0/6                                  15       14.0       140    0.2     6/6   0/6                                  7.5      13.0       130    0.6     6/6   0/6                                  Control  10         100    2.4     10/10  0/10                                ______________________________________                                         .sup.a Mice were implanted with 10.sup.6 P388 leukemia cells and              treatments with BMY41219 were begun one day later. Control mice were give     saline injections.                                                       

The present invention includes within its scope pharmaceuticalcompositions which comprise an effective tumor-inhibiting amount ofBMY-41219 of the present invention, or a pharmaceutically acceptableacid addition salt thereof, in combination with an inertpharmaceutically acceptable carrier or diluent.

According to another aspect of the invention, a method is provided fortherapeutically treating an animal (preferably mammalian) host affectedby a malignant tumor which comprises administering to such host aneffective tumor-inhibiting dose of the antibiotic of the presentinvention or a pharmaceutically acceptable acid addition salt thereof.

Examples of suitable compositions include solid compositions for oraladministration such as tablets, capsules, pills, powders and granules,liquid compositions for oral administration such as solutions,suspensions, syrups and elixirs and preparations for parenteraladministration such as sterile solutions, suspensions or emulsions. Theymay also be manufactured in the form of sterile solid compositions whichcan be dissolved in sterile water, physiological saline or some othersterile injectable medium immediately before use.

It will be appreciated that the actual preferred dosages of therebeccamycin analogs of the present invention will vary according to theparticular compound being used, the particular composition formulated,the mode of application and the particular situs, host and disease beingtreated. Many factors that modify the action of the drug will be takeninto account by those skilled in the art, e.g. age, body weight, sex,diet, time of administration, rate of excretion, condition of the host,drug combinations, reaction sensitivities and severity of the disease.Administration can be carried out continuously or periodically withinthe maximum tolerated dose. Optimal application rates for a given set ofconditions can be readily ascertained by those skilled in the art usingconventional dosage determination tests.

The present invention is illustrated by the following examples which arenot intended to be construed as limiting the scope of the invention.

EXAMPLE 1

Preparation of cryopreservative culture of Saccharothrix aerocolonigenesstrain C38,383-RK2 (ATCC 39243) .

Saccharothrix aerocolonigenes strain C38,383-RK2 was maintained as acryopreservative culture stored at -80° C. in a Revco ultralowtemperature freezer. To prepare a cryopreservative culture, strainC38,383-RK2 was transferred in test tubes on slants of yeastextract-malt extract agar supplemented with CaCO₃ which consisted of

    ______________________________________                                        dextrose       4.0 g                                                          yeast extract  4.0 g                                                          malt extract    10 g                                                          CaCO.sub.3     1.5 g                                                          agar            15 g                                                          deionized water                                                                              q.s. to 1 liter                                                ______________________________________                                    

The agar slant was incubated at 28° C. for 7-10 days. Vegetative culturewas prepared by transferring the surface growth from the slant cultureto a 500 ml Erlenmeyer flask containing 100 ml of a sterile vegetativemedium consisting of

    ______________________________________                                        Cerelose (Corn Products)                                                                             30 g                                                   Pharmamedia (Traders Oil Mill Co.)                                                                   10 g                                                   Nutrisoy (Archer Daniels Midland Co.)                                                                10 g                                                   CaCO.sub.3              3 g                                                   deionized water        q.s. to 1 liter                                        ______________________________________                                    

This vegetative culture was incubated at 28° C. for 48 hours on a rotaryshaker set at 250 rev/min. The vegetative culture was mixed with equalvolume of cryoprotective solution consisting of

    ______________________________________                                        Sucrose        100 g                                                          glycerol       200 g                                                          deionized water                                                                              q.s. to 1 liter                                                ______________________________________                                    

Four ml portions of this mixture were transferred to sterile cryogenictube (5 ml capacity, Corning) and were frozen in a dry ice-acetone bath.The frozen vegetative cultures were then stored at -80° C. in a Revcoultralow temperature freezer,

EXAMPLE 2 Preparation of Vegetative Culture of Saccharothrixaerocolonigenes strain C38,383-RK2 (ATCC 39243)

Vegetative culture was prepared by transferring 4 ml of thecryopreservative culture to a 500 ml Erlenmeyer flask containing 100 mlof a sterile vegetative medium having the same composition as thevegetative medium described in Example 1, The vegetative culture wasincubated at 28° C. for 48 hours on a rotary shaker set at 250 rev/min.

EXAMPLE 3 Preparation of BMY-41219,

Four ml of the vegetative culture of Example 2 was inoculated into 500ml Erlenmeyer flasks each containing 100 ml of a production mediumconsisting of

    ______________________________________                                        Staclipse J-UB starch (A.E. Staley)                                                                60 g                                                     Cerelose             10 g                                                     Nutrisoy             10 g                                                     Debittered Brewer's Yeast                                                                           5 g                                                     FeSO.sub.4.7H.sub.2 O                                                                               1 g                                                     (NH.sub.4).sub.2 SO.sub.4                                                                           1 g                                                     NH.sub.4 H.sub.2 PO.sub.4                                                                           1 g                                                     CaCO.sub.3           10 g                                                     deionized water      q.s. to 1 liter                                          ______________________________________                                    

The production culture was incubated at 28° C. on a rotary shaker set at250 rev/min. After 5 days of fermentation, L-tryptophan was added to theculture at a final concentration of 2 mg/ml. The culture was allowed toincubate at 28° C. and shaken at 250 rpm for additional 5 days.Production of BMY-41219 was monitored by HPLC. Optimal production of119-155 mg/ml was generally obtained at 10 days of fermentation (i.e. 5days after the addition of L-tryptophan).

EXAMPLE 4

Isolation and purification of BMY-41219:

a) Solvents and Reagents

DICALITE Speedplus Diatomite filter aid was manufactured by GREFCO, INC.Torrance, Calif. SEPHADEX LH20 was obtained from Pharmacia FineChemicals AB. Baker silica gel 7024-1 and Dimethyl Sulfoxide (DMSO) wereobtained from the J.T. Baker Chemical Company. Ammonium Acetate andAcetonitrile were HPLC grade which were obtained from Fisher.Tetrahydrofuran and Methanol were HPLC grade which were obtained fromAmerican Burdick & Jackson. Chloroform, Hexane, and Ethyl Acetate wereACS reagent grade and were not re-distilled before use. The water usedwas in house de-ionized water which was passed through a BarnsteadNanopure II water purification system.

b) General Methods

The vacuum liquid chromatography (VLC) apparatus consists of a Kontes250 ml medium porosity sintered glass funnel with side hose connectionfor vacuum and a T 24/40 joint for attachment to a receiving flask. Thefunnel was packed with dry silica which was equilibrated by drawing avolume of the least polar solvent (or solvent combination) in theelutropic series through the bed. The sample was adsorbed onto silicagel by dissolving it in an appropriate solvent, adding the adsorbentand, evaporating to dryness. The sample adsorbed silica gel was thentransferred to the funnel as a least polar solvent (or solventcombination) slurry. Vacuum elution was carried out in a step gradientfashion from least polar to most polar. The bed of silica was sucked drybetween each successive step of the gradient.

The following components were used to construct a semi-preparative highperformance liquid chromatography (HPLC) system: Waters Associates model590 Solvent Delivery System pump; Waters Associates model U6K injectorwith 2 ml loop; Whatman Partisil 10 ODS-3 (9.4mm i.d.×50 cm long)column; Knauer model 87 variable wavelength detector (320nm); Heathmodel SR-204 strip chart recorder set at 0.2 in/min. The components wereconnected with 316 (1.6mm o.d. -0.23mm i.d.) stainless steel tubing. Theeluant was pumped at 4.0 ml/min.

The following components were used to construct an analytical HPLCsystem: Waters Associates model 6000A Solvent Delivery System pump;Waters Associates U6K injector with a 2ml loop; Q-BEX ScientificIncorporated C-18 (10 um, 3.9mm i.d.×30cm) column; Hewlett PackardSpectrophotometric Detector System consisting of the HP 1040A detectorwith DPU, the HP 85B computer, the HP 9121 disk drive, the HP 7470Aplotter and, the HP THINKJET printer. The components were connected with316 (1.6mm o.d -0.23mm i.d.) stainless steel tubing. The eluant waspumped at 2.0 ml/min.

Reversed phase thin layer chromatography (TLC) was carried out onWhatman reversed phase KC18F (20×20 cm) TLC plates. 2 ul samples werespotted and plates were developed in 60% Tetrahydrofuran 40% 0.1 Mammonium acetate (V/V). The developed plates were air dried andvisualized with 366nm UV light.

c) Extraction and Purification

The whole broth (10 l) was filtered (admix) with 700 grams of Dicalitefilter aid using a 12 inch Tollhurst Laboratory Centerslung CentrifugalFilter Unit (Amtek. INC., model 1B15) lined with a sewn filter bagcoated with Dicalite filter aid. The mycelial mat was rinsed with 10liters of water, removed from bag and, stirred with 6 liters oftetrahydrofuran for 2 hours. Filtration through a 24 cm Buchner funnel(Coors no. 11) fitted with a 24 cm S & S Sharkskin paper yielded a brownorange solution which was concentrated in-vacuo to a crude residue (37.4g).

The Crude residue was tirturated with 2 liters of Chloroform andfiltered through a Kimax 2000ml-125C sintered glass funnel. Theinsoluble residue was further tirturated with 1 liter of Methanol andfiltered through the Kimax funnel. Concentration in-vacuo, of theMethanol filtrate yielded 15.2 grams of RESIDUE A.

A 250 ml sintered glass funnel was packed with 66.2 grams of silica gelwhich was equilibrated with 50% Hexane 50% Ethyl Acetate (V/V). RESIDUEA was dissolved in 50% Methanol 50% Tetrahydrofuran (V/V) to pre-adsorbit onto 25.8 grams of silica gel. The sample adsorbed silica gel wasintroduced to the equilibrated funnel as a 50% Hexane 50% Ethyl Acetate(V/V) slurry. Using pre-determined volumes and compositions of solvents,vacuum elution yielded eleven fractions as indicated below.

    ______________________________________                                        FRACTION VOLUME     ELUANT                                                    NUMBER   ELUANT     COMPOSITION (V/V)                                         ______________________________________                                        1        400 ML     50%      EtOAc  50% Hexane                                2        400 ML     60%      EtOAc  40% Hexane                                3        400 ML     70%      EtOAc  30% Hexane                                4        400 ML     80%      EtOAc  20% Hexane                                5        400 ML     90%      EtOAc  10% Hexane                                6        800 ML     100%     EtOAc                                            7        400 ML     99%      EtOAc  1% Methanol                               8        400 ML     95%      EtOAc  5% Methanol                               9        800 ML     90%      EtOAc  10% Methanol                              10       400 ML     100%     Methanol                                         11       500 ML     100%     Tetrahydrofuran                                  ______________________________________                                    

Based upon the presence of a significant orange-yellow fluorescent zone(BMY-41219) on TLC analysis, fractions 5 through 8 were combined andevaporated in-vacuo to yield 313mg of RESIDUE B.

RESIDUE B was dissolved in 3ml Tetrahydrofuran and introduced onto a2.5cm i.d.×70cm Glenco glass column which was packed with Sephadex LH20swelled in Tetrahydrofuran (Bed Dimensions were 2.5cm×68cm). Elutionwith Tetrahydrofuran was carried out at 1.0 ml/min while 250 ml eluantwas collected and discarded. Seventy 10 ml fractions were then collectedand selectively surveyed by reversed phase TLC. Fractions 13 through 19were shown by TLC to contain major amounts of BMY-41219 and weretherefore combined and evaporated in-vacuo to yield 362mg of wet RESIDUEC.

A Whatman Partisil 10 column was equilibrated with a mobile phaseconsisting of 40% Tetrahydrofuran 60% 0.1M Ammonium Acetate at 4.0ml/min. RESIDUE C. was dissolved in 2 ml 50% Tetrahydrofuran 50% mobilephase and chromatographed in six 333 uL aliquots. A single fractioncontaining one peak at about 25 minutes was collected for each run.These six fractions were combined and evaporated in-vacuo to yield 132mg BMY-41219.

What is claimed is:
 1. A process for producing the compound of theformula: ##STR5## or a pharmaceutically acceptable acid addition saltthereof, which comprises cultivating a rebeccamycin-producing strain ofSaccharothrix aerocolonigenes in an aqueous nutrient medium in thepresence of tryptophan, until a substantial amount of said compound isproduced by said organism in said culture medium and recovering saidcompound from the culture medium in a substantially pure form.
 2. Theprocess of claim 1 wherein the rebeccamycin-producing strain isSaccharothrix aerocolonigenes ATCC
 39243. 3. A method fortherapeutically treating a mammalian host affected by a tumor sensitiveto P388 leukemia which comprises administering to said host an effectivetumor-inhibiting amount of the compound as defined in claim 1.